Composition for sustained release of human growth hormone

ABSTRACT

A pharmaceutical composition for the sustained release of human growth hormone from a polymer matrix is disclosed. The pharmaceutical composition comprises a biocompatible polymer, particles of metal cation-complexed human growth hormone wherein said particles are dispersed within the biocompatible polymer and an aqueous injection vehicle.

RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. patent applicationSer. No. 09/505,508, filed Feb. 17, 2000, which is a Continuation ofU.S. patent application Ser. No. 09/252,746, filed Feb. 19, 1999 nowU.S. Pat. No. 6,051,259 which is a Continuation of U.S. patentapplication Ser. No. 09/033,193, filed Mar. 2, 1998, now U.S. Pat. No.5,891,478, which is a Continuation of U.S. patent application Ser. No.08/831,604, filed Apr. 10, 1997, now abandoned, which is a Continuationof U.S. patent application Ser. No. 08/473,544, filed Jun. 7, 1995, nowU.S. Pat. No. 5,654,010, which is a Continuation-in-Part of U.S. patentapplication Ser. No. 07/984,323, filed Dec. 2, 1992, now abandoned. Allof the above are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] Human growth hormone (hGH) is a protein secreted by the pituitarygland and which can be produced by recombinant genetic engineering. hGHwill cause growth in all bodily tissues which are capable of growth.

[0003] hGH is typically used to treat patients suffering fromhypopituitary dwarfism. Currently, aqueous hGH is administered as asubcutaneous bolus three times a week, or once daily, to patients tomaintain suitable serum levels of hGH. For patients chronicallyreceiving hGH, the frequent injections result in patient complianceproblems.

[0004] To resolve the problems associated with repetitive injections ofaqueous hGH, attempts have been made to formulate controlled releasedevices containing higher doses of hGH than a bolus injection,encapsulated within a polymeric matrix wherein the hGH would be releasedin vivo over a period of about a week or more.

[0005] However, these controlled release devices often exhibited highinitial bursts of hGH release and minimal hGH release thereafter.Further, due to the high concentration of hGH within these controlledrelease devices, the hGH molecules have tended to aggregate afterseveral days to form aggregated hGH which is immunogenic in vivo andlikely has reduced biological activity.

[0006] Therefore, a need exists for a means for sustaining the releaseof biologically active hGH in vivo without causing an immune systemresponse over the release period of the hGH.

SUMMARY OF THE INVENTION

[0007] This invention relates to a composition, and methods of formingand using said composition, for the sustained release of biologicallyactive, stabilized human growth hormone (hGH). The sustained releasecomposition of this invention comprises a polymeric matrix of abiocompatible polymer and particles of biologically active, metalcation-stabilized hGH, wherein said particles are dispersed within thebiocompatible polymer.

[0008] The method of the invention for forming a composition for thesustained release of hGH, includes dissolving a biocompatible polymer ina polymer solvent to form a polymer solution, dispersing particles ofbiologically active, stabilized hGH in the polymer solution, and thensolidifying the polymer to form a polymeric matrix containing adispersion of said hGH particles.

[0009] The method of using the sustained release composition of thepresent invention comprises providing a therapeutically effective bloodlevel of biologically active, non-aggregated human growth hormone in asubject for a sustained period by administering to the subject a dose ofsaid sustained release composition.

[0010] The advantages of this sustained release formulation for hGHinclude longer, more consistent in vivo blood levels of hGH, lowerinitial bursts of hGH, and increased therapeutic benefits by eliminatingfluctuations in serum hGH levels. The advantages also include increasedpatient compliance and acceptance by reducing the required number ofinjections. The advantages further include the ability to use smalleramounts of hGH compared to bolus injection regimen because serum hGHlevels are maintained closer to therapeutical thresholds.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention.

[0012] The human growth hormone (hGH) used in this invention isbiologically active hGH in its molecular (monomeric or non-aggregated)form. Molecular hGH is typically non-immunogenic.

[0013] Aggregated hGH may induce an immune response resulting inantibodies formed against hGH. This may compromise the efficacy oflong-term hGH therapy. Additionally, aggregated hGH may stimulate anauto-immune response to endogenous hGH.

[0014] A sustained release of biologically active, non-aggregated humangrowth hormone is a release which results in measurable serum levels ofbiologically active, monomeric hGH over a period longer than thatobtained following direct administration of aqueous hGH. It is preferredthat a sustained release be a release of hGH for a period of about aweek or more, and more preferably for a period of about two weeks ormore.

[0015] A sustained release of biologically active, non-aggregated hGHfrom a polymeric matrix can be continuous or non-continuous release withrelatively constant or varying rates of release. The continuity of hGHreleased and level of hGH released can be established by using, interalia, one or more types of polymer compositions, hGH loadings, and/orselection of excipients to produce the desired effect.

[0016] Stabilized (hGH) comprises biologically active, non-aggregatedhGH which is complexed with at least one type of multivalent metalcation, having a valency of +2 or more, from a metal cation component.Stabilized hGH in the sustained release composition of the presentinvention is in particulate form.

[0017] Suitable multivalent metal cations include metal cationscontained in biocompatible metal cation components. A metal cationcomponent is biocompatible if the cation component is non-toxic to therecipient, in the quantities used, and also presents no significantdeleterious or untoward effects on the recipient's body, such as animmunological reaction at the injection site.

[0018] Typically, the molar ratio of metal cation component to hGH, forthe metal cation stabilizing the hGH, is between about 4:1 to about100:1 and more typically about 4:1 to about 10:1.

[0019] A preferred metal cation used to stabilize hGH is Zn⁺². In a morepreferred embodiment, the molar ratio of metal cation component,containing Zn⁺² cations, to hGH is about 6:1.

[0020] The suitability of a metal cation for stabilizing hGH can bedetermined by one of ordinary skill in the art by performing a varietyof stability indicating techniques such as polyacrylamide gelelectrophoresis, isoelectric focusing, reverse phase chromatography,HPLC and potency tests on hGH lyophilized particles containing metalcations to determine the potency of the hGH after lyophilization and forthe duration of release from microparticles. In stabilized hGH, thetendency of hGH to aggregate within a microparticle during hydration invivo and/or to lose biological activity or potency due to hydration ordue to the process of forming a sustained release composition, or due tothe chemical characteristics of a sustained release composition, isreduced by complexing at least one type of metal cation with hGH priorto contacting the hGH with a polymer solution.

[0021] Stabilized hGH is typically stabilized against significantaggregation in vivo over the sustained release period. Significantaggregation is defined as an amount of aggregation resulting inaggregation of about 15% or more of the initial amount of encapsulatedhGH monomer. Preferably, aggregation is maintained below about 5% of theinitial dose of hGH monomer. More preferably, aggregation is maintainedbelow about 2% of the initial dose.

[0022] The hGH in a hGH sustained release composition can also be mixedwith other excipients, such as bulking agents or additional stabilizingagents, such as buffers to stabilize the hGH during lyophilization.

[0023] Bulking agents typically comprise inert materials. Suitablebulking agents are known to those skilled in the art.

[0024] A polymer, or polymeric matrix, suitable for the sustainedrelease composition of the present invention, must be biocompatible. Apolymer is biocompatible if the polymer, and any degradation products ofthe polymer, are non-toxic to the recipient and also present nosignificant deleterious or untoward effects on the recipient's body,such as an immunological reaction at the injection site.

[0025] The polymer of the hGH sustained release composition must also bebiodegradable. Biodegradable, as defined herein, means the compositionwill degrade or erode in vivo to form smaller chemical species.Degradation can result, for example, by enzymatic, chemical and physicalprocesses.

[0026] Suitable biocompatible, biodegradable polymers include, forexample, poly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, polycaprolactone, polycarbonates, polyesteramides,polyanhydrides, poly(amino acids), polyorthoesters, polycyanoacrylates,poly(p-dioxanone), poly(alkylene oxalate)s, biodegradable polyurethanes,blends and copolymers thereof.

[0027] Further, the terminal functionalities of the polymer can bemodified. For example, polyesters can be blocked, unblocked or a blendof blocked and unblocked polymers. A blocked polymer is as classicallydefined in the art, specifically having blocked carboxyl end groups.Generally, the blocking group is derived from the initiator of thepolymerization and is typically an alkyl group. An unblocked polymer isas classically defined in the art, specifically having free carboxyl endgroups.

[0028] Acceptable molecular weights for polymers used in this inventioncan be determined by a person of ordinary skill in the art taking intoconsideration factors such as the desired polymer degradation rate,physical properties such as mechanical strength, and rate of dissolutionof polymer in solvent. Typically, an acceptable range of molecularweights is of about 2,000 Daltons to about 2,000,000 Daltons. In apreferred embodiment, the polymer is a biodegradable polymer orcopolymer. In a more preferred embodiment, the polymer is apoly(lactide-co-glycolide) (hereinafter “PLGA”) with a lactide:glycolideratio of about 1:1 and a molecular weight of about 5,000 Daltons toabout 70,000 Daltons. In an even more preferred embodiment, themolecular weight of the PLGA used in the present invention has amolecular weight of about 6,000 to about 31,000 Daltons.

[0029] The amount of hGH, which is contained in a dose of sustainedrelease microparticles, or in an alternate sustained release device,containing biologically active, stabilized hGH particles is atherapeutically or prophylactically effective amount, which can bedetermined by a person of ordinary skill in the art taking intoconsideration factors such as body weight, condition to be treated, typeof polymer used, and release rate from the polymer.

[0030] In one embodiment, an hGH sustained release composition containsfrom about 0.01% (w/w) to about 50% (w/w) of biologically active,stabilized hGH particles. The amount of such hGH particles used willvary depending upon the desired effect of the hGH, the planned releaselevels, the times at which hGH should be released, and the time spanover which the hGH will be released. A preferred range of hGH particleloading is between about 0.1% (w/w) to about 30% (w/w) hGH particles.Amore preferred range of hGH particle loading is between about 0.1%(w/w) to about 20% (w/w) hGH particles. The most preferred loading ofthe biologically active, stabilized hGH particles is about 15% (w/w).

[0031] In another embodiment, a hGH sustained release composition alsocontains a second metal cation component, which is not contained in thestabilized hGH particles, and which is dispersed within the polymer. Thesecond metal cation component preferably contains the same species ofmetal cation, as is contained in the stabilized hGH. Alternately, thesecond metal cation component can contain one or more different speciesof metal cation.

[0032] The second metal cation component acts to modulate the release ofthe hGH from the polymeric matrix of the sustained release composition,such as by acting as a reservoir of metal cations to further lengthenthe period of time over which the hGH is stabilized by a metal cation toenhance the stability of hGH in the composition.

[0033] A metal cation component used in modulating release typicallycontains at least one type of multivalent metal cation. Examples ofsecond metal cation components suitable to modulate hGH release,include, or contain, for instance, Mg(OH)₂, MgCO₃ (such as4MgCO³.Mg(OH)₂.5H₂O), ZnCO₃ (such as 3Zn(OH)₂.2ZnCO₃), CaCO₃,Zn₃(C₆H₅O₇)₂, Mg(OAc)₂, MgSO₄, Zn(OAc)₂, ZnSO₄, ZnCl₂, MgCl₂ andMg₃(C₆H₅O₇)₂. A suitable ratio of second metal cationcomponent-to-polymer is between about 1:99 to about 1:2 by weight. Theoptimum ratio depends upon the polymer and the second metal cationcomponent utilized.

[0034] A polymeric matrix containing a dispersed metal cation componentto modulate the release of a biologically active agent from thepolymeric matrix is further described in U.S. Pat. No. 5,656,297 toBernstein et al., and co-pending PCT Patent Application PCT/US95/05511,the teachings of which are incorporated herein by reference in theirentirety.

[0035] The hGH sustained release composition of this invention can beformed into many shapes such as a film, a pellet, a cylinder, a disc ora microparticle. A microparticle, as defined herein, comprises apolymeric component having a diameter of less than about one millimeterand having stabilized hGH particles dispersed therein. A microparticlecan have a spherical, non-spherical or irregular shape. It is preferredthat a microparticle be a microsphere. Typically, the microparticle willbe of a size suitable for injection. A preferred size range formicroparticles is from about 1 to about 180 microns in diameter.

[0036] In the method of this invention for forming a composition for thesustained release of biologically active, non-aggregated hGH, a suitableamount of particles of biologically active, stabilized hGH are dispersedin a polymer solution.

[0037] A suitable polymer solution contains between about 1% (w/w) andabout 30% (w/w) of a suitable biocompatible polymer, wherein thebiocompatible polymer is typically dissolved in a suitable polymersolvent. Preferably, a polymer solution contains about 2% (w/v) to about20% (w/v) polymer. A polymer solution containing 5% to about 10% (w/w)polymer is most preferred.

[0038] A suitable polymer solvent, as defined herein, is solvent inwhich the polymer is soluble but in which the stabilized hGH particlesare substantially insoluble and non-reactuve. Examples of suitablepolymer solvents include polar organic liquids, such as methylenechloride, chloroform, ethyl acetate and acetone.

[0039] To prepare biologically active, stabilized hGH particles, hGH ismixed in a suitable aqueous solvent with at least one suitable metalcation component under pH conditions suitable for forming a complex ofmetal cation and hGH. Typically, the complexed hGH will be in the formof a cloudy precipitate, which is suspended in the solvent. However, thecomplexed hGH can also be in solution. In an even more preferredembodiment, hGH is complexed with Zn⁺².

[0040] Suitable pH conditions to form a complex of hGH typically includepH values between about 7.0 and about 7.4. Suitable pH conditions aretypically achieved through use of an aqueous buffer, such as sodiumbicarbonate, as the solvent.

[0041] Suitable solvents are those in which the hGH and the metal cationcomponent are each at least slightly soluble, such as in an aqueoussodium bicarbonate buffer. For aqueous solvents, it is preferred thatwater used be either deionized water or water-for-injection (WFI).

[0042] It is understood that the hGH can be in a solid or a dissolvedstate, prior to being contacted with the metal cation component. It isalso understood that the metal cation component can be in a solid or adissolved state, prior to being contacted with the hGH. In a preferredembodiment, a buffered aqueous solution of hGH is mixed with an aqueoussolution of the metal cation component.

[0043] Typically, the complexed hGH will be in the form of a cloudyprecipitate, which is suspended in the solvent. However, the complexedhGH can also be in solution. In an even more preferred embodiment, hGHis complexed with Zn⁺².

[0044] The complexed hGH is then dried, such as by lyophilization, toform a particulate of stabilized hGH. The complexed hGH, which issuspended or in solution, can be bulk lyophilized or can be divided intosmaller volumes which are then lyophilized. In a preferred embodiment,the complexed hGH suspension is micronized, such as by use of anultrasonic nozzle, and then lyophilized to form stabilized hGHparticles. Acceptable means to lyophilize the complexed hGH mixtureinclude those known in the art.

[0045] Preferably, particles of stabilized hGH are between about 1 toabout 6 micrometers in diameter. The hGH particles can be fragmentedseparately, as described in co-pending U.S. patent application Ser. No.08/006,682, filed Jan. 21, 1993, which describes a process for producingsmall particles of biologically active agents, which is incorporatedherein in its entirety by reference. Alternately, the hGH particles canbe fragmented after being added to a polymer solution, such as by meansof an ultrasonic probe or ultrasonic nozzle.

[0046] In another embodiment, a second metal cation component, which isnot contained in the stabilized hGH particles, is also dispersed withinthe polymer solution.

[0047] It is understood that a second metal cation component andstabilized hGH can be dispersed into a polymer solution sequentially, inreverse order, intermittently, separately or through concurrentadditions. Alternately, a polymer, a second metal cation component andstabilized hGH and can be mixed into a polymer solvent sequentially, inreverse order, intermittently, separately or through concurrentadditions.

[0048] The method for forming a composition for modulating the releaseof a biologically active agent from a biodegradable polymer is furtherdescribed in U.S. Pat. No. 5,656,297 to Bernstein et al.

[0049] In this method, the polymer solvent is then solidified to form apolymeric matrix containing a dispersion of stabilized hGH particles.

[0050] One suitable method for forming an hGH sustained releasecomposition from a polymer solution is the solvent evaporation methoddescribed in U.S. Pat. No. 3,737,337, issued to Schnoring et al., U.S.Pat. No. 3,523,906, issued to Vranchen et al., U.S. Pat. No. 3,691,090,issued to Kitajima et al., or U.S. Pat. No. 4,389,330, issued to Tice etal. Solvent evaporation is typically used as a method to form hGHsustained release microparticles.

[0051] In the solvent evaporation method, a polymer solution containinga stabilized hGH particle dispersion, is mixed in or agitated with acontinuous phase, in which the polymer solvent is partially miscible, toform an emulsion. The continuous phase is usually an aqueous solvent.Emulsifiers are often included in the continuous phase to stabilize theemulsion. The polymer solvent is then evaporated over a period ofseveral hours or more, thereby solidifying the polymer to form apolymeric matrix having a dispersion of stabilized hGH particlescontained therein.

[0052] A preferred method for forming hGH sustained releasemicroparticles from a polymer solution is described in U.S. Pat. No.5,019,400, issued to Gombotz et al., and co-pending U.S. patentapplication Ser. No. 08/443,726, filed May 18, 1995, the teachings ofwhich are incorporated herein by reference in their entirety. Thismethod of microsphere formation, as compared to other methods, such asphase separation, additionally reduces the amount of hGH required toproduce a sustained release composition with a specific hGH content.

[0053] In this method, the polymer solution, containing the stabilizedhGH particle dispersion, is processed to create droplets, wherein atleast a significant portion of the droplets contain polymer solution andthe stabilized hGH particles. These droplets are then frozen by meanssuitable to form microparticles. Examples of means for processing thepolymer solution dispersion to form droplets include directing thedispersion through an ultrasonic nozzle, pressure nozzle, Rayleigh jet,or by other known means for creating droplets from a solution.

[0054] Means suitable for freezing droplets to form microparticlesinclude directing the droplets into or near a liquified gas, such asliquid argon and liquid nitrogen to form frozen microdroplets which arethen separated from the liquid gas. The frozen microdroplets are thenexposed to a liquid non-solvent, such as ethanol, or ethanol mixed withhexane or pentane.

[0055] The solvent in the frozen microdroplets is extracted as a solidand/or liquid into the non-solvent to form stabilized hGH containingmicroparticles. Mixing ethanol with other non-solvents, such as hexaneor pentane, can increase the rate of solvent extraction, above thatachieved by ethanol alone, from certain polymers, such aspoly(lactide-co-glycolide) polymers.

[0056] A wide range of sizes of hGH sustained release microparticles canbe made by varying the droplet size, for example, by changing theultrasonic nozzle diameter. If very large microparticles are desired,the microparticles can be extruded through a syringe directly into thecold liquid. Increasing the viscosity of the polymer solution can alsoincrease microparticle size. The size of the microparticles can beproduced by this process, for example microparticles ranging fromgreater than about 1000 to about 1 micrometers in diameter.

[0057] Yet another method of forming an hGH sustained releasecomposition, from a polymer solution, includes film casting, such as ina mold, to form a film or a shape. For instance, after putting thepolymer solution containing a dispersion of stabilized hGH particlesinto a mold, the polymer solvent is then removed by means known in theart, or the temperature of the polymer solution is reduced, until a filmor shape, with a consistent dry weight, is obtained. Film casting of apolymer solution, containing a biologically active agent, is furtherdescribed in U.S. Pat. No. 5,656,297 to Bernstein et al., the teachingsof which are incorporated herein by reference in their entirety.

[0058] It is believed that the release of the hGH can occur by twodifferent mechanisms. The hGH can be released by diffusion throughaqueous filled channels generated in the polymeric matrix, such as bythe dissolution of the hGH or by voids created by the removal of thepolymer's solvent during the synthesis of the sustained releasecomposition.

[0059] A second mechanism is the release of hGH due to degradation ofthe polymer. The rate of degradation can be controlled by changingpolymer properties that influence the rate of hydration of the polymer.These properties include, for instance, the ratio of different monomers,such as lactide and glycolide, comprising a polymer; the use of theL-isomer of a monomer instead of a racemic mixture; and the molecularweight of the polymer. These properties can affect hydrophilicity andcrystallinity, which control the rate of hydration of the polymer.Hydrophilic excipients such as salts, carbohydrates and surfactants canalso be incorporated to increase hydration and which can alter the rateof erosion of the polymer.

[0060] By altering the properties of the polymer, the contributions ofdiffusion and/or polymer degradation to hGH release can be controlled.For example, increasing the glycolide content of apoly(lactide-co-glycolide) polymer and decreasing the molecular weightof the polymer can enhance the hydrolysis of the polymer and thus,provides an increased hGH release from polymer erosion.

[0061] In addition, the rate of polymer hydrolysis is increased innon-neutral pH's. Therefore, an acidic or a basic excipient can be addedto the polymer solution, used to form the microsphere, to alter thepolymer erosion rate.

[0062] The composition of this invention can be administered to a human,or other animal, by injection, implantation (e.g, subcutaneously,intramuscularly, intraperitoneally, intracranially, intravaginally andintradermally), administration to mucosal membranes (e.g., intranasallyor by means of a suppository), or in situ delivery (e.g. by enema oraerosol spray) to provide the desired dosage of hGH based on the knownparameters for treatment with hGH of the various medical conditions.

[0063] The invention will now be further and specifically described bythe following examples.

EXAMPLE 1

[0064] Formation of Zn⁺²-Stabilized hGH

[0065] Human growth hormone (hGH), whose DNA sequence is described inU.S. Pat. No. 4,898,830, issued to Goeddel et al., was used in thisExample. The human growth hormone was stabilized by forming insolublecomplexes with zinc.

[0066] The hGH was dissolved in samples of a 4 mM sodium bicarbonatebuffer (pH 7.2) to form hGH solutions with a concentrations between 0.1and 0.5 mM hGH. A 0.9 mM Zn⁺² solution was prepared from deionized waterand zinc acetate dihydrate and then was added to the hGH solutions toform Zn⁺²-hGH complex. The pH of the Zn⁺²-hGH complex was then adjustedto between 7.0 and 7.4 by adding 1% acetic acid. A cloudy suspendedprecipitate, comprising Zn⁺²-stabilized hGH formed.

[0067] The suspension of Zn⁺²-stabilized hGH was then micronized usingan ultrasonic nozzle (Type V1A; Sonics and Materials, Danbury, Conn.)and sprayed into a polypropylene tub (17 cm diameter and 8 cm deep)containing liquid nitrogen to form frozen particles. The polypropylenetub was then placed into a −80 ° C. freezer until the liquid nitrogenevaporated. The frozen particles, which contained Zn⁻²-stabilized hGH,were then lyophilized to form Zn⁺²-stabilized hGH particles.

EXAMPLE 2

[0068] Preparation of PLGA Microspheres Containing Biologically Active,Aggregation-Stabilized hGH

[0069] Microspheres containing Zn⁺²-stabilized human growth hormone(hGH) were prepared from hydrophilic poly(lactide-co-glycolide) polymerRG502H having free carboxyl end groups (hereinafter “unblocked-PLGA”)(50:50 PLGA, 9,300 Daltons; Boehringer Ingelheim Chemicals, Inc.) or amore hydrophobic PLGA polymer having blocked carboxyl end groups(hereinafter “blocked-PLGA”) (50:50 PLGA, 10,000 Daltons; Lot #115-56-1,Birmingham Polymers, Inc., Birmingham, Ala.).

[0070] The polymer was dissolved in methylene chloride at roomtemperature. The lyophilized hGH particles were added to the polymersolution and zinc carbonate was also added. The mixture was thensonicated to give a homogeneous suspension. The suspension was atomizedthrough a sonicating nozzle on to a bed of frozen ethanol, overlaid withliquid nitrogen. The vessel containing the microspheres was stored at−80° C. to extract the methylene chloride and then freeze-dried to givea free-flowing powder.

EXAMPLE 3

[0071] Analysis of Encapsulated hGH Protein

[0072] The integrity of encapsulated hGH was determined by dissolvingunhydrated microspheres into methylene chloride and acetone, collectingthe protein, freeze-drying and re-constituting in HEPES buffercontaining 10 mM EDTA. Appropriate controls were run to ensure that theextraction process did not affect the integrity of the protein.

[0073] The integrity of the encapsulated hGH was analyzed by measuringthe percent of hGH monomer contained in the hGH after encapsulationthrough size exclusion chromatography (SEC).

[0074] The results of SEC analyses of the hGH integrity of hGH sustainedrelease microspheres are provided below. Formulation (polymer; % ZincCarbonate) % Monomer (SEC) 31 K unblocked; 6% ZnCO3 98.6 31 K unblocked;6% ZnCO3 99.2 31 K unblocked; 3% ZnCO3 97.7 31 K unblocked; 3% ZnCO397.8 31 K unblocked; 1% ZnCO3 97.6 31 K unblocked; 0% ZnCO3 97.8 31 Kunblocked; 0% ZnCO3 97.1 10 K blocked; 1% ZnCO3 98.2 10 K blocked; 1%ZnCO3 98.4  8 K unblocked; 0% ZnCO3 98.5 10 K blocked; 1% ZnCO3 98.4

[0075] The results showed that the encapsulation process did not causeaggregation of the protein. The yield percent protein recovered by theextraction procedure (relative to the amount measured by nitrogencontent of the microspheres) ranged from about 40 to 98%. Thevariability is thought to be associated with loss of material during thetransfer steps in the procedure and the extraction procedure is beingmodified to increase protein recovery.

EXAMPLE 4

[0076] Determination of the Effect of Zinc Carbonate on In vitro ReleaseKinetics

[0077] The microspheres were formed as described in Example 2 andcontained 15% w/w hGH (6:1 Zn:hGH protein complex); 0%, 1%, 6%, 10% or20% w/w zinc carbonate; and poly(lactide-co-glycolide) polymer.

[0078] In vitro release kinetics of the hGH sustained releasemicrosphere formulations containing various concentrations of zinccarbonate were determined by suspending an aliquot (10 mg) of each typeof microsphere in different 1.5 ml samples of HEPES buffer (50 mM Hepes,10 mM KCl, 0.1% NaN3) pH 7.2 and then incubating at 37° C. The amount ofprotein released was quantitated by sampling the buffer at 1, 3, 7, 10,14, 21, 28 days after incubation and replenishing with fresh bufferafter each sampling.

[0079] A curve of cumulative percent released (relative to initial hGHcontent in the starting mass of microspheres) versus time was plotted.Released protein samples from each time point were assayed for hGHmonomer content by size exclusion chromatography.

[0080] Zinc carbonate is thought to act as a reservoir of zinc ions sothat the formation of the Zn-hGH complex is favored and dissociationinto soluble hGH disfavored. Because the aqueous solubility of zinccarbonate is low, the release of zinc ions from the reservoir is slowthus modulating the solubility of the protein.

[0081] The analysis found that in the absence of zinc carbonate, therate of release of the encapsulated hGH was very rapid and all theprotein was released in a very short period.

EXAMPLE 5

[0082] Assay for hGH After in Vivo Degradation of Blocked-PLGAZn⁺²-Stabilized hGH Microspheres

[0083] Microspheres of blocked-PLGA, containing 15% w/w Zn⁺²-stabilizedhGH and 0%, 6%, 10% or 20% ZnCO₃ were formed by the method of Example 2.Groups of test rats were injected subcutaneously with 50 mg samples ofthe different hGH microspheres. The rats were sacrificed after 60 daysand the skin sample were excised from the injection sites. The excisedskin samples were placed in 10% Neutral Buffered Formalin for at least24 hours. They were then trimmed with a razor blade to remove excessskin and placed in PBS.

[0084] Tissue samples were processed by Pathology Associates, Inc.(Frederick, Md.). The skin samples were embedded in glycomethacrylate,sectioned and assayed for the presence of hGH using aHistoScan/LymphoScan Staining Kit (Product #24-408M; Accurate Chemical &Scientific Corp., Westbury, N.Y.) according to the manufacturer'sinstructions. Tissue samples were scored for the presence or absence ofstaining which was indicative of the presence or absence of hGH in thesample.

[0085] All skin samples, associated with hGH microsphere injections,tested positive for the presence of hGH thus indicating that theblocked-PLGA microspheres still contained hGH after 60 days in vivo.

[0086] The method described in Example 2 was used to form microspheresby encapsulating 0% or 15% w/w hGH, in the form of Zn:hGH complex, andalso 0%, 1% or 6% w/w ZnCO₃ salt, within blocked-PLGA and withinunblocked-PLGA.

[0087] In vivo degradation of unblocked-PLGA microspheres versusblocked-PLGA microspheres were compared by injecting samples ofmicrospheres into rats and then analyzing the microspheres remaining atthe injection site at various times post-injection. Three rats wereassayed at each time point for each microsphere sample. On the day ofadministration of the microspheres, 750 μl of vehicle (3% carboxymethylcellulose (low viscosity) and 1% Tween-20 in saline) was added to vialscontaining 50±1 mg of microspheres. Immediately, the vials were shakenvigorously to form a suspension which was then aspirated into a 1.0 ccsyringe without a needle.

[0088] Rats (Sprague-Dawley males) were anesthetized with a halothaneand oxygen mixture. The injection sites (intrascapular region) wereshaven and marked with a permanent tatoo to provide for the preciseexcision of skin at the sampling time points. Each rat was injected withan entire vial of microspheres using 18 to 21 gauge needles.

[0089] On designated days (days 15, 30, 59 and 90 post-injection foranimals receiving blocked-PLGA microspheres, or days 7, 14, 21, 28 and45 post-injection for animals receiving unblocked-PLGA microspheres) therats were sacrificed by asphyxiation with CO₂ gas and the skin at theinjection sites (including microspheres) was excised. Since themicrospheres tended to clump at the injection sites, the presence orabsence of microspheres was determined visually.

[0090] The visual inspections found that the unblocked-PLGA microspheresdegraded substantially faster than the blocked-PLGA microspheres, andthat the addition of ZnCO₃ to the blocked-PLGA substantially slowedpolymeric degradation. For example, in the rats injected withunblocked-PLGA microspheres containing 0% hGH and 0% or 1% ZnCO₃, nomicrospheres were visible on day 21. In addition, for rats injected withblocked-PLGA microspheres containing 0% hGH and 0% ZnCO₃, a fewmicrospheres were visible on day 60 and none were visible on day 90.Furthermore, for rats injected with blocked-PLGA microspheres containing0% or 15% hGH and 6% ZnCO₃, microspheres were visible on day 90.

EXAMPLE 6

[0091] In Vivo Pharmnacokinetic Studies of hGH Sustained ReleaseMicrospheres in Rats

[0092] Studies were conducted in rats to screen various hGH microsphereformulations, determine pharmacokinetic parameters following intravenous(IV), subcutaneous (SC) and SC osmotic pump (Alzet) administration ofhGH, and to evaluate serum profiles and in vivo release-rate of varioushGH microsphere formulations.

[0093] Sprague-Dawley rats were divided into groups of three each,randomized by body weight, and one hGH microsphere formulation wasadministered to each group. Rats were injected subcutaneously withapproximately 7.5 mg of hGH in 50 mg of one type of the differentmicrospheres, suspended in 0.75 ml of an aqueous injection vehicle. Thevehicle composition was 3% CMC (low viscosity), 1% Polysorbate 20, in0.9% NaCl. The microsphere dose delivered was determined indirectly byweighing the residual dose in the injection vial and correcting forresidual injection vehicle. The hGH dose was then computed from theprotein loading of the microspheres determined by nitrogen analysis.

[0094] Blood samples were collected at pre-determined intervals for upto 30 days after injection. Blood samples of 250 μl were collectedduring the first 24 hours and at least 400 μl at time points after 24hours. Blood samples were clotted and hGH concentrations in serum weredetermined using a radio-immuno assay. A radio-immunoassay (RIA) kitfrom ICN was validated and used to determine the hGH levels in ratserum.

[0095] For the determination of pharmacokinetic parameters, hGH insaline was administered to rats by subcutaneous bolus injection,intravenously or delivered via an osmotic pump (Alzet Model 2ML4) whichwas implanted subcutaneously.

[0096] Three groups of rats received single subcutaneous injections ofhGH in 0.9% NaCl at 0.5 or 7.5 mg/kg at a dose volume of 1.0 ml/kg andtwo groups received single intravenous bolus injections of hGH in 0.9%NaCl solution at about 1.0 mg and 5.0 mg of hGH per kg rat with a dosevolume of 1.0 ml/kg. For the Alzet pump study, rats were divided intofour groups of three rats each, randomized by body weight and dosed withabout 20 mg/ml and 40 mg/ml hGH in 0.9% saline solution loaded intopumps (Alzet Model 2002, 200 μl, 14 days release), and with about 4mg/ml and 12 mg/ml hGH in 0.9% saline solution loaded into pumps (AlzetModel 2ML4, 2ml, 28 days release). Expected release rates from the pumpscorrespond to about 2% and 4 to 6% of the ProLease hGH dose (about 15mg/kg) per day, respectively. The Alzet pumps were implantedsubcutaneously in the inter-scapular region after soaking for 1-2minutes in sterile saline.

[0097] The formulations of hGH sustained release microspheres,synthesized as described in Example 2 contained 15% w/w hGH complexedwith Zn in a ratio of 6:1 Zn:hGH; 0%, 1%, 3% or 6% w/w zinc carbonate;and 8K unblocked PLGA, 10K blocked PLGA or 31K unblocked PLGA.

[0098] To evaluate the various hGH sustained release formulations, Cmax,Cd5 and Cmax/Cd5 were the in vivo indices used, where Cmax is themaximum serum concentration observed, and Cd5 is the serum concentrationat day 5 which should approximate the steady state concentration. Theresults were as follows: Burst % Monomer C day 5 Formulation in vitro(%) Day 7 Cmax (ng/ml) (ng/ml) Cmax/Css 8 K PLGA 22.0 ± 0.9 99.3* 323.3± 98.6 20.4 ± 14.2 19.5 ± 10.6 unblocked 0% ZnCO3 8 K PLGA 16.4 ± 1.697.3* 309.0 ± 67.1 20.4 ± 14.2 39.5 ± 17.7 unblocked 1% ZnCO3 8 K PLGA15.9 ± 6.9 98.7  670.5 ± 244.4 9.0 ± 4.2 44.8 ± 22.6 unblocked 3% ZnCO38 K PLGA 17.6 ± 2.7 99.3 358.0 ± 58.9 18.8 ± 14.7 42.4 ± 6.8  unblocked6% ZnCO3 31 K PLGA 12.3 ± 1.1 98.2   592 ± 318.2 4.5 ± 1.5 132.5 ± 47.9 unblocked 0% ZnCO3 31 K PLGA 11.4 ± 1.3 98.8 432.7 ± 91.6 5.1 ± 0.3 84.1± 14.9 unblocked 3% ZnCO3 31 K PLGA  7.9 ± 1.9 99.4  643.6 ± 203.9 8.0 ±2.6 93.3 ± 62.0 unblocked 3% ZnCO3 31 K PLGA 15.8 ± 0.5 99.8 1691.8 ±340.0 6.6 ± 0.8 262.2 ± 83.5  unblocked 6% ZnCO3 10 K PLGA 12.7 ± 0.199.3  615.9 ± 384.3 4.5 ± 1.0 155.0 ± 126.8 blocked 1% ZnCO3 10 K PLGA18.1 ± 3.2 99.6 1053.2 ± 293.3 3.6 ± 0.8 291.7 ± 71.1  blocked 3% ZnCO310 K PLGA  9.9 ± 1.4 99.0 1743.5 ± 428.4 4.9 ± 2.7 516.1 ± 361.6 blocked6% ZnCO3

[0099] The results of the screening showed that the two unblockedpolymers (8K and 31K) had different in vivo release kinetics compared tothe original formulation, which used blocked 10K PLGA and 6% w/w zinccarbonate. Cmax values were generally lower with the unblocked polymerformulations than with the lead formulation which suggested that the invivo burst may be lower with the unblocked polymer formulations. Theburst was defined as the percent of hGH released in the first 24 hoursafter injection. The in vitro burst values were between 8-22%. The zinccarbonate content of the formulations did not appear to have an effecton the burst or the in vitro release profile.

[0100] The serum concentrations between days 4 and 6 were maintained ata fairly constant level above baseline (or the pre-bleed levels) withthe unblocked polymer formulations, while serum concentrations with theblocked formulations, at the same time points were close to the baselinelevels. The in vitro release data for up to 7 days showed that thereleased hGH protein was monomeric. Useful data could not be obtainedbeyond day 6 because of anti-hGH antibody formulation in the rats.

EXAMPLE 7

[0101] Rhesus Monkey Pharmacokinetics Study

[0102] The objective of this primate study was to evaluate thepharmacokinetic profiles of different hGH sustained release formulationsas compared to more traditional methods of administering hGH (e.g.,bolus sc injections, daily sc injections and sc injection combined withthe use of an osmotic pump) and to determine which hGH sustained releaseformulation gave the optimal hGH blood concentration profile.

[0103] The formulations for the hGH sustained release microspherestested were 1) 15% hGH (complexed with Zn at a 6:1 Zn:hGH ratio), 6% w/wzinc carbonate and 10K blocked PLGA; 2) 15% hGH (complexed with Zn at a6:1 Zn:hGH ratio), 1% w/w zinc carbonate and 8K unblocked PLGA (“RG502H”PLGA polymer); and 3) 15% hGH (complexed with Zn at a 6:1 Zn:hGH ratio),1% w/w zinc carbonate and 31K unblocked PLGA (“RG503H” PLGA polymer).

[0104] There were four monkeys per group and each animal received asingle subcutaneous injection into the dorsal cervical region on Day 1.A dose of 160 mg of hGH sustained release microspheres (24 mg of hGH)was administered to each monkey in 1.2 ml of injection vehicle through a20 gauge needle. The injection vehicle was an aqueous vehicle containing3% w/v Carboxymethyl Cellulose (sodium salt), 1% v/v Tween 20(Polysorbate 20) and 0.9% sodium chloride.

[0105] The hGH dose was intended to provide measurable hGH serumconcentrations for pharmacokinetic analysis. To obtain pharmacokineticparameters additional study groups of four monkeys each were included,specifically 1) a single subcutaneous injection (24 mg hGH), 2) dailysubcutaneous injections (24 mg/28 days=0.86 mg hGH/day), 3) asubcutaneous injection (3.6 mg hGH) combined with an Alzet osmotic pump(20.4 mg hGH)(total dose of 24 mg hGH), and 4) a subcutaneous injectionof the injection vehicle as a control (only used 3 monkeys for thevehicle control group).

[0106] Blood samples were collected at the following times for hGH,IGF1, IGFBP3 and anti-hGH antibody analyses: −7, −5, −3, pre-dose and,0.5, 1, 2, 3, 5, 8, 10, 12, 24, 28,32 and 48 hours, 5, 4, 6, 8, 11, 14,17, 20, 23, 26, 29, 32, 25, 28, 41, 44, 47, 50, 53, 56 days post-dose.

[0107] The concentrations of IGF-1 and hGH in the serum were thenmeasured. An IRMA kit from RADIM (distributed by: Wein Laboratories,P.O. Box 227, Succasunna, N.J.) was used to quantify hGH in monkeyserum. The IRMA assay had a limit of quantification in PBS buffer of 0.1ng/mL and in pooled juvenile rhesus monkey serum of 1.5 ng/mL with abasal GH level of about 4 ng/mL.

[0108] The IRMA assay was validated over the concentration range 1.5-75ng/mL for pooled juvenile rhesus monkey serum. The measurement precisionand accuracy are within the range of ±10%.

[0109] The results showed that the hGH sustained release microsphereswere releasing significant, sustained levels of hGH over a one monthperiod while the subcutaneous injections were not able to maintain thesame serum levels.

[0110] The IGF-1 serum profile showed that serum IGF-1 concentrationswere elevated above the baseline values between days 2 and 29 afteradministering the microparticles. This shows that enough hGH was beingreleased from the hGH sustained release microspheres to cause apharmacodynamic effect. This also indicates that the hGH released wasbiologically active which suggest that the encapsulation process had notadversely affected the biopotency of hGH.

[0111] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

What is claimed is:
 1. A pharmaceutical composition for the sustainedrelease of human growth hormone from a polymer matrix, comprising: a) abiocompatible polymer; b) particles of metal cation-complexed humangrowth hormone, wherein said particles are dispersed within thebiocompatible polymer; and c) an aqueous injection vehicle.